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Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma

Abstract

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

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Figure 1: Visualization of HBV integration breakpoints in the HCC and HBV genomes.
Figure 2: Overall statistics for HBV integration events in HCC tumors.
Figure 3: Mapping of HBV breakpoint integration sites.
Figure 4: Influence of HBV integration on gene expression in HCC.
Figure 5: Clinical correlation analysis of HBV integration in HCC.

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NCBI Reference Sequence

References

  1. 1

    Center, M.M. & Jemal, A. International trends in liver cancer incidence rates. Cancer Epidemiol. Biomarkers Prev. 20, 2362–2368 (2011).

    Article  PubMed  Google Scholar 

  2. 2

    But, D.Y., Lai, C.L. & Yuen, M.F. Natural history of hepatitis-related hepatocellular carcinoma. World J. Gastroenterol. 14, 1652–1656 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  3. 3

    Ishikawa, T. Clinical features of hepatitis B virus–related hepatocellular carcinoma. World J. Gastroenterol. 16, 2463–2467 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    Bréchot, C., Gozuacik, D., Murakami, Y. & Paterlini-Brechot, P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin. Cancer Biol. 10, 211–231 (2000).

    Article  PubMed  Google Scholar 

  5. 5

    Zucman-Rossi, J. & Laurent-Puig, P. Genetic diversity of hepatocellular carcinomas and its potential impact on targeted therapies. Pharmacogenomics 8, 997–1003 (2007).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Gehring, A.J. et al. Profile of tumor antigen–specific CD8 T cells in patients with hepatitis B virus–related hepatocellular carcinoma. Gastroenterology 137, 682–690 (2009).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Shafritz, D.A., Shouval, D., Sherman, H.I., Hadziyannis, S.J. & Kew, M.C. Integration of hepatitis B virus DNA into the genome of liver cells in chronic liver disease and hepatocellular carcinoma. Studies in percutaneous liver biopsies and post-mortem tissue specimens. N. Engl. J. Med. 305, 1067–1073 (1981).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Koshy, R. et al. Integration of hepatitis B virus DNA: evidence for integration in the single-stranded gap. Cell 34, 215–223 (1983).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Brechot, C., Pourcel, C., Louise, A., Rain, B. & Tiollais, P. Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 286, 533–535 (1980).

    CAS  Article  Google Scholar 

  10. 10

    Chakraborty, P.R., Ruiz-Opazo, N., Shouval, D. & Shafritz, D.A. Identification of integrated hepatitis B virus DNA and expression of viral RNA in an HBsAg-producing human hepatocellular carcinoma cell line. Nature 286, 531–533 (1980).

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Bonilla Guerrero, R. & Roberts, L.R. The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma. J. Hepatol. 42, 760–777 (2005).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Murakami, Y. et al. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 54, 1162–1168 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Paterlini-Bréchot, P. et al. Hepatitis B virus–related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 22, 3911–3916 (2003).

    Article  PubMed  Google Scholar 

  14. 14

    Gozuacik, D. et al. Identification of human cancer-related genes by naturally occurring Hepatitis B Virus DNA tagging. Oncogene 20, 6233–6240 (2001).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Saigo, K. et al. Integration of hepatitis B virus DNA into the myeloid/lymphoid or mixed-lineage leukemia (MLL4) gene and rearrangements of MLL4 in human hepatocellular carcinoma. Hum. Mutat. 29, 703–708 (2008).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Ferber, M.J. et al. Integrations of the hepatitis B virus (HBV) and human papillomavirus (HPV) into the human telomerase reverse transcriptase (hTERT) gene in liver and cervical cancers. Oncogene 22, 3813–3820 (2003).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Neuveut, C., Wei, Y. & Buendia, M.A. Mechanisms of HBV-related hepatocarcinogenesis. J. Hepatol. 52, 594–604 (2010).

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Totoki, Y. et al. High-resolution characterization of a hepatocellular carcinoma genome. Nat. Genet. 43, 464–469 (2011).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Li, M. et al. Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma. Nat. Genet. 43, 828–829 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Furuta, T. et al. Clinicopathologic features of hepatocellular carcinoma in young patients. Cancer 66, 2395–2398 (1990).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Kim, J.H. et al. Clinical features and prognosis of hepatocellular carcinoma in young patients from a hepatitis B–endemic area. J. Gastroenterol. Hepatol. 21, 588–594 (2006).

    Article  PubMed  Google Scholar 

  22. 22

    Hao, K. et al. Predicting prognosis in hepatocellular carcinoma after curative surgery with common clinicopathologic parameters. BMC Cancer 9, 389 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Xu, X. et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol. 29, 735–741 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Chen, Y. et al. Complete genome sequence of hepatitis B virus (HBV) from a patient with fulminant hepatitis without precore and core promoter mutations: comparison with HBV from a patient with acute hepatitis infected from the same infectious source. J. Hepatol. 38, 84–90 (2003).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Lamb, J.R. et al. Predictive genes in adjacent normal tissue are preferentially altered by sCNV during tumorigenesis in liver cancer and may rate limiting. PLoS ONE 6, e20090 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Chiang, D.Y. et al. High-resolution mapping of copy-number alterations with massively parallel sequencing. Nat. Methods 6, 99–103 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Mermel, C.H. et al. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol. 12, R41 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We gratefully acknowledge Y.-K. Mak and the clinical team of the Division of Hepatobiliary and Pancreatic Surgery (HBP) at Queen Mary Hospital. This study was funded by the Asian Cancer Research Group (ACRG), a not-for-profit organization formed by Eli Lilly, Merck and Pfizer. We thank S. Friend and G. Jin for initiating the establishment of ACRG. We are grateful to former and present members of ACRG, especially K. Blanchard, Y. Turpaz, J. Sedgwick, G. Tucker-Kellogg, G. Gilliland, P. Shaw, N. Gibson and S. Adams.

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Correspondence to Christoph Reinhard or Jun Wang or John M Luk.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note (PDF 1422 kb)

Supplementary Table 2

Summary of sequencing depth and coverage of 88 (TU/AN) pairs of HCC. (XLS 46 kb)

Supplementary Table 3

Characterization of 399 HBV integration breakpoints identified in HCC. (XLS 139 kb)

Supplementary Table 4

Validation result for the HBV integration sites. (XLS 40 kb)

Supplementary Table 5

Correlation analysis of HBV integration (cutoff = 1 to 6) with various clinic-pathological parameters of HCC patients (n = 83). (XLSX 13 kb)

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Sung, WK., Zheng, H., Li, S. et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nat Genet 44, 765–769 (2012). https://doi.org/10.1038/ng.2295

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